The long-term, major objective of this research is to understand how guanosine tetraphosphate (ppGpp), a general signal molecule, or "alarmone," for amino acid deprivation in bacteria interacts at the molecular level to activate transcription of the histidine biosynthetic operon (and, by analogy, many other genes). Guanosine tetraphosphate appears to be part of a super-control system that adjusts many cellular processes in response to the cell's need for amino acids. The specific goals are to identify the target DNA sequence in the promoter region of the Salmonella typhimurium histidine operon at which the regulatory effect of ppGpp is exerted and to characterize the protein component(s) with which the molecule interacts to regulate gene expression. The approach being used to analyze the mechanism of ppGpp regulation at the his promoter combines molecular-genetic and biochemical methods of analysis. The his promoter is being dissected through analysis of spontaneous mutations selected for decreased promoter activity, as well as through analysis of oligonucleotide-directed, site-specific mutations, and other site- specific mutations, isolated in vitro. Effects of the promoter mutations on ppGpp-mediated regulation will be assessed using cell-free protein synthesis in vitro, and with physiological experiments in vivo employing amino acid shiftdown conditions. Regulatory mutants having lesions in genes coding for potential protein factors with which ppGpp interacts will be analyzed using these methods as well. Unravelling the mechanisms by which gene expression is regulated is the key to understanding the intricate phenomena of differentiation and development. This knowledge may also help to understand the causation of genetic and regulatory disorders such as cancer. A major impetus for this research on bacterial gene regulatory mechanisms is the anticipation that generalizations arising from these studies will apply to all living cells.